Arbuscular mycorrhiza-upregulated PtPIP1;2 facilitates H2O2 transport to enhance drought tolerance in trifoliate orange

Abstract Arbuscular mycorrhizal fungi (AMF) can enhance drought tolerance of host plants; however, the underlying molecular mechanisms remain unclear. Here, PtPIP1;2, a plasma membrane intrinsic protein gene strongly induced by AMF (Funneliformis mosseae) colonization under drought stress, was identified. Subcellular localization confirmed that PtPIP1;2 targets the plasma membrane. To investigate its function, PtPIP1;2-overexpressing (OE) hairy root lines were generated in trifoliate orange. Under drought stress, OE plants exhibited better growth performance, greater root biomass and surface area, showed alleviated inhibition of photosynthesis and photosystem II activity, and reduced oxidative damage. Notably, OE plants showed lower root hydrogen peroxide (H2O2) content but significantly enhanced H2O2 efflux. Heterologous expression assays revealed that when expressed alone in Xenopus oocytes, PtPIP1;2 did not confer detectable water permeability; however, it markedly enhanced yeast sensitivity to exogenous H2O2, indicating that PtPIP1;2 contributes to H2O2 transport-associated drought tolerance. Consistently, OE plants maintained a favorable hormonal balance (higher abscisic acid content together with partially restored trans-zeatin, indole-3-acetic acid, and zeatin levels) and showed upregulated expression of stress-responsive genes involved in calcium signaling (PtCDPK10), MAPK cascades (PtMAPK), dehydration protection (PtLEA7), polyamine synthesis (PtADC), and antioxidant enzymes (PtSOD and PtPOD). Collectively, these findings suggest that PtPIP1;2 enhances drought tolerance by facilitating transmembrane H2O2 transport, which is associated with improved reactive oxygen species turnover, a more favorable hormonal balance, and the activation of downstream defense networks. These findings reveal a potential mechanism by which an AMF-inducible aquaporin may act as a redox-regulatory hub to protect plants against drought, and identify PtPIP1;2 as a promising target for breeding drought-tolerant citrus rootstocks.